The information of inclusion bodies is a serious problem in bacterial expression systems. Quite often, these insoluble aggregates represent most or all of the recombinant protein expressed. It has been observed that E. coli protease(s) degrade(s) the inclusion body proteins during denaturation and refolding, a process necessary to recover active, soluble protein. In addition, proteins expressed in the cytosol of E. coli are frequently degraded after cell lysis and during purification procedures. I have identified the protease involved in the degradation of Torpedo californica creatine kinase (TCK) inclusion bodies in E. coli upon denaturation as OmpT, an outer membrane protease in E. coli. The proteolytic activity can be completely removed by detergent extraction of the inclusion bodies, and the extract gives identical degradation products of TCK by SDS Polyacrylamide Gel Electrophoresis (SDS/PAGE) showing that the extract contains the proteolytic activity. The activity of OmpT under denaturing conditions necessary to refold proteins has been further characterized using both TCK and rabbit muscle creatine kinase (RMCK) as substrates using SDS/PAGE. N-terminal sequence analysis of degradation fragments of both TCK and RMCK show that the protease has a specificity for argarg and lys-lys bonds. The protease OmpT is also specific for dibasic residues. The protease activity has been shown to be completely inhibited by ZnC12, CuC12 and benzamidine which are specific inhibitors of OmpT. In addition, PMSF and EDTA which are known to inhibit all other membrane proteases in E. coli do not inhibit this activity. No proteolysis was observed in an extract from an OmpT deletion mutant. This mutant was also used to overexpress OmpT, and the proteolytic degradation pattern appears identical to that observed in the TCK inclusion body system. I have partially purified OmpT from the extract from the TCK inclusion bodies and verified the identity of this protease activity as OmpT by N-terminal sequence analysis. In order to determine the susceptibility of native proteins to this protease activity, accesible sites on the protein surface can be determined by analysing proteins with available crystal structures using MIDAS. Proteins with significant sequence homology to proteins with crystal structure data could also be modeled using MIDAS. I was also able to identify other related proteins to OmpT by the use of primary sequence homology. The OmpT protease appears to be a member of a potentially novel class of proteases based on its lack of sequence homology to other known proteases. Solving the protease problem for recovery of soluble, active proteins from inclusion bodies in bacterial expression systems is of critical importance. In addition, soluble expressed proteins are susceptible to E. coli proteases. This project would provide the necessary tools for recovering full-length soluble proteins by designating appropriate inhibitors and mutant cell lines for bacterial expression systems.